Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels.
A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.
Jahnen
W
W
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, (Melbourne Branch), Parkville, Victoria, Australia.
Ward
L D
LD
Reid
G E
GE
Moritz
R L
RL
Simpson
R J
RJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010940:Plant Proteins / Q000302:isolation & purification; D010947:Plants, Toxic; D011506:Proteins / Q000302:isolation & purification*; D014026:Nicotiana
D010446:Peptide Fragments; D010940:Plant Proteins; D011506:Proteins; D003488:Cyanogen Bromide